The goal of this grant proposal is to initiate a new area of research that will investigate novel pathways of epithelium formation. Epithelia perform a vital function to keep tissues, organs and cells in distinct topological compartments. For example, sheets of epithelia line organs, epithelial tubes transport liquids and gases throughout the body. Classic experiments established the paradigm that the adhesion protein E-cadherin and its partners 1-catenin and 2-catenin initiate polarity during epithelium formation. More recent work using 3D culture systems has implicated integrins and phosphoinositides as polarizing cues to create epithelia. These studies relied on tissue culture models that can be induced to form epithelial structures in vitro or on genetic organisms, predominantly Drosophila. Our aim is to complement the classical approaches with a new genetic system for epithelium development. The C. elegans arcade cells undergo a mesenchymal to epithelial transition, and the resulting epithelial tube links the gut to the exterior epidermis. The impetus for our new project is the observation that C. elegans generates this epithelium (and its other epithelia) independent of known epithelial regulators including cadherins, catenins or integrins. We hypothesize that the C. elegans arcade cells rely on an alternative pathway to build epithelia, one that is amenable to genetic analyses and in vivo imaging. We will analyze the MKLP kinesin zen-4 during epithelium formation. We previously showed that inactivation of zen-4 blocks epithelium formation of the arcade cells, the strongest epithelial defect yet observed in C. elegans. We will analyze ZEN-4 and its role in epithelia, as an entry point towards understanding non-canonical epithelium formation. These experiments will establish the C. elegans arcade cells as a model for epithelium formation, and will set the stage for understanding non-canonical epithelium formation. PUBLIC HEALTH RELEVANCE: Epithelia are composed of highly polarized, adherent cells with an asymmetric distribution of proteins, lipids and organelles within the cytoplasm and at the cell surface. Epithelia perform a vital function to keep tissues, organs and cells in distinct topological compartments. This proposal seeks to establish a new model system to study epithelium formation, one that will uncover non-classical pathways for polarity and epithelialization. )